Field of the Invention
The present invention provides a method for the analysis (including detection) of a compound having an amino group, such as amino acid and peptide, by using mass spectrometry with a high sensitivity analytical reagent that enhances the selectivity of the compound. The present invention also provides an analytical (labeling) reagent that may be used for the aforementioned method; a method for the labeling a compound having an amino group; and a novel carbamate compound as the analytical reagent.
Discussion of the Background
Compounds with amino groups—including amino acids, peptides and others—play an important role in living body. In view of this importance, a critical demand exists in the fields of medicine, pharmaceutical science, agriculture, biochemistry and clinical chemistry to quantitate the relative abundance thereof. In particular, there is a critical demand for a selective and highly sensitive quantitative determination method for particularly amino acids, especially where the sample amount and the concentration of the analyte is varied and impurities are likely to be present. A typical example of where the demand exists is when amino acids of metabolites in cells are to be quantified. Moreover, by improving the selectivity and sensitivity of the quantitative method less sample amount is needed, thus reducing the physical and mental burden of the person to be tested.
Most amino acids have very low ultraviolet absorption, fluorescence and/or electrochemical response. Heretofore, to enhance the analytical sensitivity, a method where an amino group is labeled with compound, chromophore or fluorophore having a large ultraviolet absorptivity and is detected by ultraviolet, visible light or fluorescence has been usually used. In regard to a representative ultraviolet labeling reagent, phenylisothiocyanate (PITC) [cf. Cohen, S. A. and Strydom, D. J., 174, 1 (1988)] has been employed while ninhydrin has been widely known as a visible labeling reagent. Both of these reagents are commercially available. In regard to a fluorescence labeling reagent, o-phthalaldehyde (OPA) [cf. Roth, M., Anal. Chem. 43, 880 (1971)], dansyl chloride (Dansyl-Cl) [cf. Seiler, N., Methods Biochem. Anal., 18, 259 (1970)], 4-fluoro-7-nitrobenzofurazan (NBD-F) [cf. lmai, K. and Watanabe, Y., Anal. Chim. Acta, 130, 377 (1981)], etc. have also been reported and are commercially available.
In the aforementioned analytical methods, the limit of the analytical sensitivity for ultraviolet/visible light is typically about 1 pmol, while amount of amino acid which can be detectable by using fluorescence is about 100 fmol. Thus, in the field of biochemistry and clinical chemistry, there has been a demand for further improvement in the sensitivity. Further, where labeling substances that have an absorption in an ultraviolet region (PITC; 254 nm) or labeling substance having excitation and fluorescence wavelength that are in relatively short wavelength (OPA: (λ)ex=340 to 345 nm, (λ)em=455 nm; Dansyl-CI: (λ)ex=255 nm, (λ)em=470 nm), accurate quantification is frustrated by the presence of impurities.
Usually, to achieve a quantitative analytical result an excessive reagent quantity must be added to the analyte during the labeling reaction leading to a need for additional purification. If the purification step is omitted, the quantitative results are often difficult to accurately interpret since for many labeling substances (e.g., PITC, Dansyl-Cl and NBD-F) the reagent or a hydrolysate thereof has strong ultraviolet absorption and fluorescence. Therefore, the reagent or a hydrolysate thereof is a big impediment in the analysis.
6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) [cf. Iwaki, K., Yoshida, S., Nimura, N., Kinoshita, T., Takeda, K., and Ogura, H., Chromatographia, 23, 899 (1987)] has been also developed as a reagent to label an amino group of an amino acid to enable fluorescence detection. However, this reagent is plagued by the same problems discussed above in that its excitation wavelength and fluorescence wavelength are in a short wavelength range of (λ)ex=245 nm and (λ)em=395 nm, respectively. Therefore, AQC is apt to be affected by impurities. Moreover, the reagent and a hydrolysate thereof also have fluorescence properties.
As stated above, when the aforementioned labeling reagents are used a purification method where the labeled compound is separated by a liquid chromatography (hereinafter, referred to as HPLC) has been performed to analyze the amino acid. In the HPLC, there are many cases where retention ability of the substance to be analyzed in a column varies by slight changes in the environment or, in other words, by slight changes in the composition of the mobile phase or by changes in column temperature. Since detection is conducted by absorption or fluorescence of the labeling reagent, each amino acid should be identified by means of column-retention ability. When an analyte having unpredictable retention ability is detected, the fact that the substance has an amino group can be noted but any other information concerning the structure can not be ascertained.
Accordingly, the current state of the art in which analysis of a labeled compound with amino group by means of ultraviolet ray, visible ray, or fluorescence has another problem in the selectivity. Therefore, the present invention seeks to meet the critical demand for a selective and highly sensitive quantitative determination method for a compound having an amino group (e.g., amino acids), especially where the sample amount and the concentration of the analyte is varied and impurities are likely to be present.